The C2BBe1 (brush border expressing) cell line was cloned in 1988 from the Caco-2 cell line by limiting dilution; the clone was selected on the basis of morphological homogeneity and exclusive apical villin localization; C2BBe1 cells form a polarized monolayer with an apical brush border (BB) morphologically comparable to that of the human colon; isolated BB contained the microvillar proteins villin, fimbrin, sucrase-isomaltase, BB myosin-1 and the terminal web proteins fodrin and myosin II ; the cells express substantial levels of BB myosin I similar to that of the human enterocyte; although clonal, and far more homogeneous than the parental Caco-2 cell line with respect to BB expression, these cells are still heterogenous for microvillar length, microvillar aggregation, and levels of expression of certain BB proteins.

C2BBe1 cells are sensitive to any imbalance in the media pH. If you are using a DMEM medium formulation containing 3.7 g/L sodium bicarbonate, C2BBe1 cells must be incubated in a 95% air; 10% carbon dioxide (CO2) environment . The increased CO2 is necessary when using 3.7 g/L bicarbonate in order for the medium to be buffered properly, or these cells will not attach well. If you use the recommended ATCC DMEM medium (Catalog No. 30-2002) which contains only 1.5 g/L sodium bicarbonate, this medium is formulated for use with 5% CO2. The basal medium should be supplemented with 10% non heat inactivated Fetal Bovine Serum (FBS) (ATCC® 30-2020™) and 0.01 mg/mL human transferrin (Sigma T-5391). Any floating cells may be viable and should be retained by gentle centrifugation and added back to the adherent population at the time of medium renewal or subculture.

For more information, please contact Cobioer (4008-750-250).

II. Handling Procedure for Flask Cultures

The flask was seeded with cells grown and completely filled with medium at Cobioer to prevent loss of cells during shipping.

1.Upon receipt visually examine the culture for macroscopic evidence of any microbial contamination.

Using an inverted microscope (preferably equipped with phasecontrast optics), carefully check for any evidence of microbial contamination. Also check to determine if the majority of cells are still attached to the bottom of the flask? during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable).

2.If the cells are still attached, aseptically remove all but 5 to 10 mL of the shipping medium. The shipping medium can be saved for reuse. Incubate the cells at 37°C in a 5% CO2 in air atmosphere until they are ready to be subcultured.

3.If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 1000rpm for 5 to 10 minutes. Remove shipping medium and save. Resuspend the pelleted cells in 10 mL of this medium and add to 25 cm2 flask. Incubate at 37°C in a 5% CO2 in air atmosphere until cells are ready to be subcultured.

III. Subculturing Procedure

Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

3.Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (depend on the batch of trypsin).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

3.Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 1 to 10 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.